Fig 1.
PI3K-C2α knockdown decreases autophagy.
(A-E) U2OS cells stably expressing ptfLC3B were transfected with siRNAs directed against each of the eight PI3K isoforms for 48 hours. Cells were imaged with a 60× oil objective by fluorescent microscopy. Scale bars represent 10 μm. (A) Using a control siRNA, cells were treated with either vehicle or rapamycin for 6 hours to induce autophagy. (B-D) siRNA directed against each of the Class I PI3K isoforms PIK3CA, PIK3CB, PIK3CG, PIK3CD (B), PIK3C2B or PIK3C2G (C), and PIK3C2A or PIK3C3 (D) and rapamycin treated for 6 hours. (E) Quantification of the number of autophagic puncta per cell in (A-D). Data represent means of n ≥ 25 cells with standard error of the mean. Unpaired t test, comparing experimental to rapamycin control. *denotes p < 0.05, ***denotes p < 0.001.
Fig 2.
PI3K-C2α is a positive regulator of autophagy.
(A-D) The number of GFP-LC3-II puncta that accumulated in the presence (light gray circles) or absence (dark gray circles) of BafA1 was plotted (t = 0 through t = 70 min). Values on the vertical axis represent mean numbers of puncta with adjustment such that the value at t = 0 is 0. Means were adjusted by subtracting the mean number of vesicles at t = 0. (A) Vehicle treated control siRNA cells, and (B) rapamycin induced autophagy in control siRNA conditions after rapamycin addition at time t = 0. (C, D) PIK3C2A siRNA knockdown (48 hours) and cells treated with vehicle control (C) or rapamycin (D). Bars represent standard deviations.
Fig 3.
PI3K-C2α knockdown inhibits sustained autophagy and results in the formation of lipid droplets.
(A) U2OS cells stably expressing ptfLC3B were transfected with siRNAs directed to PIK3C2A, PIK3C3, and ULK1 for 48 hours. Live-cell imaging was carried out for six continuous hours after addition of rapamycin (50 nM) to measure changes in GFP-LC3 puncta accumulation. (B) U2OS cells were transfected with siRNAs directed to PIK3C2A, PIK3C3, and ULK1 for 48 hours, and then treated with rapamycin (6 hours) to induce autophagy in the absence or presence of BafA1 (90 mins). ATG8 isoforms (LC3A, LC3B, and GABARAP) and p62 (SQSTM1) were examined to detect changes in autophagy. (C) U2OS cells stably expressing EGFP-LC3B were transfected with siRNAs directed to PIK3C2A for 48 hours. After 24 hours, siRNA-resistant wild-type protein (WT-PI3K-C2α) or siRNA-resistant kinase-dead protein (KD-PI3K-C2α) was transfected. After an additional 24 hours, cells were treated with rapamycin for 6 hours and number of puncta per cell counted from exogenous PI3K-C2α expressing cells. Representative images are shown in S3 Fig. Data represent means of n ≥ 25 cells with standard error of the mean. Unpaired t test, comparing experimental to control. ***denotes p < 0.001. (D) Loss of PI3K-C2α increases lipid droplets (LD) as measured by transmission electron microscopy. Insets are 5× magnifications.
Fig 4.
PI3K-C2α colocalizes with markers of early endocytosis.
mCherry-PI3K-C2α expressing U2OS cell line was fixed and stained with primary antibodies against clathrin (A), EEA1 (B), RAB5 (C), RAB11 (D), LC3B (E), and LAMP2 (F). Secondary antibody (green) marks primary antibody staining. Cells were imaged with a 60× oil objective by confocal microscopy. Boxes are 5× magnifications of insets. Scale bar 10 μm.
Fig 5.
PI3K-C2α elutes and interacts with endocytic and autophagosomal markers.
(A) U2OS fractions (P2, P3, and S3) from differential centrifugation (see S6 Fig) and OptiPrep gradient pellets (Fractions 5 and 6) were probed for markers of endocytosis and autophagy. (B) V5-PI3K-C2α or V5-PI3K-C3 were immunoprecipitated from 293FT cells and lysates probed for markers of endocytosis and autophagy. (C) Wild-Type (WT) or Kinase-Dead [64] V5-PI3K-C2α were immunoprecipitated from 293FT cells and lysates probed for markers of endocytosis and autophagy. (B, C) Whole cell lysates (WCL) were probed with the indicated antibodies as controls.
Fig 6.
PI3K-C2α or ATG9 knockdown results in transferrin accumulation at the recycling endosome.
U2OS cells were transfected with either control siRNAs or siRNAs directed to PIK3C2A, ATG9A/B, or PIK3C3 for 48 hours. Following rapamycin treatment (6 hours), cells were incubated with Texas Red-conjugated transferrin, washed with fresh media, and returned to 37°C. Cells were stained with antibodies for endogenous clathrin (A), EEA1 (B), and RAB11 (C). Secondary antibody (green) marks primary antibody staining. Cells were imaged using confocal with a 60× oil objective. Scale bar shows 10 μm. (D) Percent colocalization of endogenous proteins with Texas Red-transferrin vesicles. Unpaired t test comparing experimental and control. *denotes p < 0.05, ***denotes p < 0.001.
Fig 7.
Proposed model for how PI3K-C2α may facilitate the integration of endocytic membrane sources into the autophagy pathway through the recycling endosome.
Model highlighting PI3K-C2α localization in the endocytic and autophagy pathways. Abbreviations: CCP: clathrin-coated pit; EE: early endosome; LE: late endosome; MVB: multi-vesicular body; RE: recycling endosome.